Access to cellular components such as nucleic acids is imperative to a variety of molecular biology methodologies. Such methodologies include nucleic acid sequencing, direct detection of particular nucleic acid sequences by nucleic acid hybridization and nucleic acid sequence amplification techniques.
The preparation and purification of high-purity double-stranded (ds) plasmid DNA, single-stranded (ss) phage DNA, chromosomal DNA, agarose gel-purified DNA fragments and RNA is of critical importance in molecular biology. Ideally, a method for purifying nucleic acids should be simple, rapid and require little. if any, additional sample manipulation. Nucleic acids rendered by such a method should be immediately amenable to transformation, restriction analysis, litigation or sequencing. A method with all of these features would be extremely attractive in the automation of nucleic acid sample preparation, a goal of research and diagnostic laboratories.
Typically, the preparation of plasmid DNA from crude alcohol precipitates is laborious, most often utilizing CsCl gradients, gel filtration, ion exchange chromatography, or RNase, proteinase K and repeated alcohol precipitation steps. These methods also require considerable downstream sample preparation to remove CsCl and other salts, ethidium bromide and alcohol. Similar arguments extend when using any of these methods for purifying DNA fragments. A further problem with these methods is that small, negatively-charged cellular components can co-purify with the DNA. Thus, the DNA can have an undesirable level of contamination.
Nucleic acids can also be purified using solid phases. Conventional solid phase extraction techniques have utilized surfaces which either (1) fail to attract and hold sufficient quantities of nucleic acid molecules because of surface design to permit easy recovery of the nucleic acid molecules during elution, or (2) excessively adhere nucleic acid molecules to the surface, thereby hindering recovery of the nucleic acid molecules during elution. Conventional metal surfaces which cause these problems when utilized in solid phase extraction include certain silica surfaces such as glass and Celite. Adequate binding of nucleic acids to these types of surfaces can be achieved only by utilizing high concentrations of chaotropes or alcohols which are generally toxic, caustic, and/or expensive. For example, it is known that DNA will bind to crushed glass powders and to glass fiber filters in the presence of chaotropes. The chaotropic ions typically are washed away with alcohol, and the DNAs are eluted with low-salt solutions or water. Importantly, RNA and protein do not bind. However, a serious drawback in the use of crushed glass powder is that its binding capacity is low. In addition. glass powders often suffer from inconsistent recovery, incompatibility with borate buffers and a tendency to nick large DNAs. Similarly, glass fiber filters provide a nonporous surface with low DNA binding capacity. Other silicas, such as silica gel and glass beads, are not suitable for DNA binding and recovery. Currently, the solid phase of choice for solid phase extraction of DNA is Celite such as found in Prep-A-Gene(trademark) by Bio-Rad Laboratories. As with the crushed glass powders, high concentrations of chaotropes are required for adequate binding of the DNA to the Celite.
However, the hydration of silica substances has, in some instances resulted in elimination of the need for such high concentrations of chaotropes to elute bound DNA from the silica substance as taught in references such as EP 0 512 767, EP 0 585 660, U.S. Pat. No. 5,674,997 and EP 0 832 897.
There are numerous protocols for purifying DNA. For example, U.S. Pat. No. 4,923,978 discloses a process for purifying DNA in which a solution of protein and DNA is passed over a hydroxylated support and the protein is bound and the DNA is eluted. U.S. Pat. No. 4,935,342 discloses purification of DNA by selective binding of DNA to anion exchangers and subsequent elution. U.S. Pat. No. 4,946,952 discloses DNA isolation by precipitation with water-soluble ketones. A DNA purification procedure using chaotropes and dialyzed DNA is disclosed in U.S. Pat. No. 4,900,677.
Diatoms have also been utilized for purification of nucleic acids as evidenced by U.S. Pat. No. 5,234,809 to Boom et al. and U.S. Pat. No. 5,075,430 to Little et al.
Yet a further technique utilized for purification of nucleic acids is binding to specifically adapted paramagnetic particles. Examples of such techniques may be found in references such as European Specification EP 0 446 260 B1 and U.S. Pat. No. 5,512,439 (Homes et al.) which describe monodisperse, superparamagnetic particles having a particle diameter standard deviation of less than 5%. Each particle carries a plurality of molecules of an oligonucleotide, with each oligonucleotide having a section serving as a probe for a target nucleic acid molecule of interest.
U.S. Pat. No. 4,672,040 (Josephson) and U.S. Pat. No. 4,695,393 (Whitehead et al.) describe magnetically responsive particles for use in systems to separate certain molecules. The particles have a metal oxide core surrounded by a stable silicone coating to which organic and/or biological molecules may be coupled.
U.S. Pat. No. 3,970,518 (Giaever) describes a method of sorting and separating a select cell population from a mixed cell population. The method utilizes small magnetic particles which are coated with an antibody to the select cell populations.
U.S. Pat. No. 4,141,687 (Forrest et al.) describes an automatic apparatus and method to assay fluid samples. The apparatus utilizes a particulate material with a reagent bound thereto. The particulate material is magnetic, and the reagent is a substance which takes part in a reaction in the reaction mixture.
U.S. Pat. No. 4,230,685 (Senyei et al.) describes a method for magnetic separation of cells. The method utilizes magnetically-responsive microspheres which are coated with staphylococcal Protein A to which is bound antibody.
U.S. Pat. No. 4,774,265 (Ugelstad et al.) describes a process for preparing magnetic polymer particles. The particles are compact or porous polymer particles treated with a solution of iron salts.
U.S. Pat. No. 5,232,782 (Charmot) describes magnetizable xe2x80x9ccore-shellxe2x80x9d microspheres which have a core of a magnetizable filler and a shell of crosslinked organopolysiloxane.
U.S. Pat. No. 5,395,688 (Wang et al.) describes magnetically responsive fluorescent polymer particles which have a polymeric core coated evenly with a layer of polymer containing magnetically responsive metal oxide.
U.S. Pat. No. 5,491,068 and U.S. Pat. No. 5,695,946 (Benjamin et al.) describe an assay method for detecting the presence of bacteria using magnetic beads with specific antibodies immobilized thereon.
U.S. Pat. No. 5,536,644 (Ullman et al.) describes a particle separation method. The method utilizes magnetic particles with surface functional groups, and optionally, an additional surface coating.
European Patent Specification EP 0 444 120 B1 (Homes et al.) describes a method for detection of target RNA or DNA. The method utilizes magnetic particles carrying a single stranded 5xe2x80x2-attached DNA probe capable of binding the target RNA or DNA.
International Publication No. WO 96/18731 (Deggerdal et al.) describes a method for isolating nucleic acid from a sample using a particulate solid support and an anionic detergent.
U.S. patent No. 5,705,628 (Hawkins) describes a method for DNA purification and isolation using magnetic particles with functional group-coated surfaces.
In order to provide a more effective and efficient technique for the purification and manipulation of nucleic acids, the present invention relates to a composition useful for reversible binding of a nucleic acid molecule. The composition includes a paramagnetic particle in an acidic environment. The invention also includes such a composition packaged as a kit, as well as methods utilizing such a composition to reversibly bind a nucleic acid molecule.
The present invention relates to unique compositions of matter. More specifically, the composition of matter is a paramagnetic particle in an acidic solution, that is, a solution having a pH of less than about 7.0.
The Applicants found that when in an acidic environment, paramagnetic particles will reversibly bind nucleic acid molecules without the necessity of an anionic detergent as taught in International Publication No. WO 96/18731. Although not desiring to be bound by a particular theory, the Applicants believe that an acidic environment increases the electropositive nature of the iron portion of the molecules, and thus increases the binding of the molecules to the electronegative phosphate portion of a nucleic acid molecule.
As used herein, the term paramagnetic particles means particles which are capable of having a magnetic moment imparted to them when placed in a magnetic field. Therefore, such paramagnetic particles, when in such a magnetic field, are movable under the action of such a field. Such movement is useful for moving bound nucleic acid molecules for different aspects of a sample processing protocol or other manipulations. Thus, nucleic acid molecules bound to the paramagnetic particles can be moved to different areas for exposure to different reagents and/or conditions with minimal direct contact due to the application of magnetic force.
The Applicants have found that paramagnetic particles useful in the present invention need not be complicated structures. Thus, iron particles are useful in the present invention, and the iron may be an iron oxide of forms such as ferric hydroxide and ferrosoferric oxide, which have low solubility in an aqueous environment. Other iron particles such as iron sulfide and iron chloride may also be suitable for binding and extracting nucleic acids using the conditions described herein.
Similarly, the shape of the paramagnetic particles is not critical to the present invention. Thus, the paramagnetic particles may be of various shapes, including for example, spheres, cubes. oval, capsule-shaped, tablet-shaped, non-descript random shapes, etc., and may be of uniform shape or non-uniform shapes. Whatever the shape of a paramagnetic particle, its diameter at its widest point is generally in the range of from about 0.5 xcexcm to about 20 xcexcm.
The acidic environment in which the paramagnetic particles effectively, reversibly bind nucleic acid molecules can be provided through a variety of means. For example, the paramagnetic particles can be added to an acidic solution, or an acidic solution may be added to the particles. Alternatively a solution or environment in which the paramagnetic particles are located can be acidified by addition of an acidifying agent such as hydrochloric acid, sulfuric acid, acetic acid and citric acid.
Provided that the environment in which the paramagnetic particles are located is of a pH less than about 7.0, the particles will reversibly bind nucleic acid molecules. Furthermore, the Applicants have found that the nucleic acid binding capacity of the paramagnetic particles increases as the pH decreases.
The acidic environment for the paramagnetic particles of the present invention is believed to allow for elimination of the need for detergents as taught in certain references such as International Publication No. WO 96/18731. Without desiring to be held to a particular theory, the Applicant believes that detergents are not necessary for the present invention, because the acidic solution of the present invention promotes the binding of electropositive paramagnetic particles to electronegative nucleic acid molecules in preference to other substances in a sample such as nucleic acid hybridization and amplification inhibitors. In contrast, the utilization of detergents as taught in references such as International Publication No. WO 96/18731 is solubilize nucleic acid hybridization and amplification inhibitors in order that such inhibitors do not interfere with binding of nucleic acid molecules to paramagnetic particles.
As stated above, in an acidic environment, electropositive paramagnetic particles, such as ferric oxide particles, will bind electronegative nucleic acid molecules. Thus, other materials in the environment, such as inhibitors of nucleic acid hybridization and amplification can be separated from the bound nucleic acid molecules. Such separation can be accomplished by means known to those skilled in the art, such as centrifugation, filtering or application of magnetic force.
The bound nucleic acid molecules can then be eluted into an appropriate buffer for further manipulation, such as hybridization or amplification reactions. Such elution can be accomplished by heating the environment of the particles with bound nucleic acids and/or raising the pH of such environment. Agents which can be used to aid the elution of nucleic acid from paramagnetic particles include basic solutions such as potassium hydroxide, sodium hydroxide or any compound which will increase the pH of the environment to an extent sufficient that electronegative nucleic acid is displaced from the particles.
The following examples illustrate specific embodiments of the invention described in this document. As would be apparent to skilled artisans, various changes and modifications are possible and are contemplated within the scope of the invention described.